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The activation of the Mirror Neuron System (MNS) has been described to reflect visible movements, but not postural, non-visible, adaptations that accompany the observed movements. Since any motor act is the result of a well-tailored dialogue between these two components, we decided to investigate whether a motor resonance to nonvisible postural adaptations could be detected. Possible changes in soleus corticospinal excitability were investigated by eliciting the H-reflex during the observation of three videos, corresponding to three distinct experimental conditions: 'Chest pass', 'Standing' and 'Sitting', and comparing its size with that measured during observation of a control videoclip (a landscape). In the observed experimental conditions, the Soleus muscle has different postural roles: a dynamic role in postural adaptations during the Chest pass; a static role while Standing still; no role while Sitting. The H-reflex amplitude was significantly enhanced in the 'Chest pass' condition compared to the 'Sitting' and 'Standing' conditions. No significant difference was found between 'Sitting' and 'Standing' conditions. The increased corticospinal excitability of the Soleus during the 'Chest pass' condition suggests that the mirror mechanisms produce a resonance to postural components of an observed action, although they may not be visible. This observation highlights the fact that mirror mechanisms echo non intentional movements as well and points to a novel possible role of mirror neurons in motor recovery.
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Neuronas Espejo , Electromiografía , Músculo Esquelético/fisiología , Movimiento , Reflejo H/fisiologíaRESUMEN
Intellectual Disability is a common and heterogeneous disorder characterized by limitations in intellectual functioning and adaptive behaviour, whose molecular mechanisms remain largely unknown. Among the numerous genes found to be involved in the pathogenesis of intellectual disability, 10% are located on the X-chromosome. We identified a missense mutation (c.236 C > G; p.S79W) in the SYN1 gene coding for synapsin I in the MRX50 family, affected by non-syndromic X-linked intellectual disability. Synapsin I is a neuronal phosphoprotein involved in the regulation of neurotransmitter release and neuronal development. Several mutations in SYN1 have been identified in patients affected by epilepsy and/or autism. The S79W mutation segregates with the disease in the MRX50 family and all affected members display intellectual disability as sole clinical manifestation. At the protein level, the S79W Synapsin I mutation is located in the region of the B-domain involved in recognition of highly curved membranes. Expression of human S79W Synapsin I in Syn1 knockout hippocampal neurons causes aberrant accumulation of small clear vesicles in the soma, increased clustering of synaptic vesicles at presynaptic terminals and increased frequency of excitatory spontaneous release events. In addition, the presence of S79W Synapsin I strongly reduces the mobility of synaptic vesicles, with possible implications for the regulation of neurotransmitter release and synaptic plasticity. These results implicate SYN1 in the pathogenesis of non-syndromic intellectual disability, showing that alterations of synaptic vesicle trafficking are one possible cause of this disease, and suggest that distinct mutations in SYN1 may lead to distinct brain pathologies.
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Discapacidad Intelectual Ligada al Cromosoma X/genética , Mutación Missense , Sinapsinas/genética , Vesículas Sinápticas/genética , Animales , Secuencia de Bases , Humanos , Discapacidad Intelectual Ligada al Cromosoma X/metabolismo , Ratones , Ratones Noqueados , Mutación , Neurogénesis/genética , Plasticidad Neuronal/genética , Neuronas/metabolismo , Linaje , Terminales Presinápticos/metabolismo , Cultivo Primario de Células , Transporte de Proteínas , Sinapsinas/metabolismo , Transmisión Sináptica/genética , Vesículas Sinápticas/metabolismoRESUMEN
The classical view of multiple sclerosis (MS) pathogenesis states that inflammation-mediated demyelination is responsible for neuronal damage and loss. However, recent findings show that impairment of neuronal functions and demyelination can be independent events, suggesting the coexistence of other pathogenic mechanisms. Due to the inflammatory milieu, subtle alterations in synaptic function occur, which are probably at the basis of the early cognitive decline that often precedes the neurodegenerative phases in MS patients. In particular, it has been reported that inflammation enhances excitatory synaptic transmission while it decreases GABAergic transmission in vitro and ex vivo. This evidence points to the idea that an excitation/inhibition imbalance occurs in the inflamed MS brain, even though the exact molecular mechanisms leading to this synaptic dysfunction are as yet not completely clear. Along this line, we observed that acute treatment of primary hippocampal neurons in culture with pro-inflammatory cytokines leads to an increased phosphorylation of synapsin I (SynI) by ERK1/2 kinase and to an increase in the frequency of spontaneous synaptic vesicle release events, which is prevented by SynI deletion. In vivo, the ablation of SynI expression is protective in terms of disease progression and neuronal damage in the experimental autoimmune encephalomyelitis mouse model of MS. Our results point to a possible key role in MS pathogenesis of the neuronal protein SynI, a regulator of excitation/inhibition balance in neuronal networks.
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Encefalomielitis Autoinmune Experimental/metabolismo , Sinapsinas/metabolismo , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Hipocampo/metabolismo , Inflamación/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Esclerosis Múltiple/patología , Neuronas/metabolismo , Fármacos Neuroprotectores/metabolismo , Fosforilación , Sinapsis/metabolismo , Sinapsinas/genética , Vesículas Sinápticas/metabolismoRESUMEN
Identity-differentiating self from external reality-and agency-being the author of one's acts-are generally considered intrinsic properties of awareness and looked at as mental constructs generated by consciousness. Here a different view is proposed. All physiological systems display complex time-dependent regulations to adapt or anticipate external changes. To interact with rapid changes, an animal needs a nervous system capable of modelling and predicting (not simply representing) it. Different algorithms must be employed to predict the momentary location of an object based on sensory information (received with a delay), or to design in advance and direct the trajectory of movement. Thus, the temporal dynamics of external events and action must be handled in differential ways, thereby generating the distinction between self and non-self ("identity") as an intrinsic computational construct in neuronal elaboration. Handling time is not what neurons are designed for. Neuronal circuits are based on parallel processing: each bit of information diverges on many neurons, each of which combines it with many other data. Spike firing reports the likelihood that the specific pattern the neuron is designed to respond to is present in the incoming data. This organization seems designed to process synchronous datasets. However, since neural networks can introduce delays in processing, time sequences can be transformed into simultaneous patterns and analysed as such. This way predictive algorithms can be implemented, and continually improved through neuronal plasticity. To successfully interact with the external reality, the nervous system must model and predict, but also differentially handle perceptual functions or motor activity, by putting in register information that becomes available at different time moments. Also, to learn through positive/negative reinforcement, modelling must establish a causal relation between motor control and its consequences: the contrast between phase lag in perception and phase lead (and control) in motor programming produces the emergence of identity (discerning self from surrounding) and agency (control on actions) as necessary computational constructs to model reality. This does not require any form of awareness. In a brain, capable of producing awareness, these constructs may evolve from mere computational requirements into mental (conscious) constructs.
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The precise subcellular organization of synaptic vesicles (SVs) at presynaptic sites allows for rapid and spatially restricted exocytotic release of neurotransmitter. The synapsins (Syns) are a family of presynaptic proteins that control the availability of SVs for exocytosis by reversibly tethering them to each other and to the actin cytoskeleton in a phosphorylation-dependent manner. Syn ablation leads to reduction in the density of SV proteins in nerve terminals and increased synaptic fatigue under high-frequency stimulation, accompanied by the development of an epileptic phenotype. We analyzed cultured neurons from wild-type and Syn I,II,III(-/-) triple knock-out (TKO) mice and found that SVs were severely dispersed in the absence of Syns. Vesicle dispersion did not affect the readily releasable pool of SVs, whereas the total number of SVs was considerably reduced at synapses of TKO mice. Interestingly, dispersion apparently involved exocytosis-competent SVs as well; it was not affected by stimulation but was reversed by chronic neuronal activity blockade. Altogether, these findings indicate that Syns are essential to maintain the dynamic structural organization of synapses and the size of the reserve pool of SVs during intense SV recycling, whereas an additional Syn-independent mechanism, whose molecular substrate remains to be clarified, targets SVs to synaptic boutons at rest and might be outpaced by activity.
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Sinapsinas/deficiencia , Transmisión Sináptica/fisiología , Vesículas Sinápticas/fisiología , Vesículas Sinápticas/ultraestructura , Animales , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Cultivo Primario de Células , Ratas , Ratas Sprague-Dawley , Sinapsinas/genética , Sinapsinas/fisiología , Transmisión Sináptica/genéticaRESUMEN
Several theories of consciousness (ToC) have been proposed, but it is hard to integrate them into a consensus theory. Each theory has its merits, in dealing with some aspects of the question, but the terminology is inconsistent, each ToC aims at answering a different question, and there is not even a reasonable agreement about what 'consciousness' is in the first place. Some common implicit assumptions, and the way some critical words - such as 'sensation', 'perception', 'neural correlate of consciousness' (NCC) - are thought to relate to consciousness, have introduced a series of misconceptions that make it difficult to pinpoint what consciousness consists in and how it arises in the brain. The purpose of this contribution is twofold: firstly, to discern the various steps that lead from the detection of a stimulus to a conscious experience, by redefining terms such as sensation and perception with an adequate operative meaning; secondly, to emphasize the inevitable contribution of emotions and the active role of imagination in this process. The diffuse view, for the layperson but among scientists as well, is that the brain produces an internal 'representation' of the external reality and of oneself. This tends to consign one to a Cartesian perspective, i.e., the idea that some entity must be there to witness and interpret such representation. This approach splits the conscious experience into brain activity, which generates a (possible) content of consciousness (still unconscious), and a vaguely defined entity or process that 'generates' consciousness and injects (or sheds the light of) consciousness onto the content of brain activity. This way, however, we learn nothing about how such consciousness would arise. We propose here that consciousness is the function that generates a subjectively relevant and emotionally coloured internal image of the experience one is living. In this process, endogenous, spontaneous activity (imaginative activity, consisting in recalling and reviving memories, prefiguring consequences, analysing conjectures) produces many vague and ambiguous hints, rich of symbolic links, which compete in giving rise to an implicit, emotionally characterized, and semantically pleiotropic, internal experience. Cognitive elaboration may extract from this a defined and univocal, complete and consistent, explicit experience, that can be verbally reported ('what it is like to...').
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Encéfalo , Estado de Conciencia , Imaginación , Emociones , Recuerdo MentalRESUMEN
The brain cannot stop elaborating information. While the circuitries implied in processing sensory information, and those involved in programming and producing movements, have been extensively studied and characterized, what circuits elicit and sustain the endogenous activity (which might be referred to as imaginative activity) has not been clarified to a similar extent. The two areas which have been investigated most intensely are visual and motor imagery. Visual imagery mostly involves the same areas as visual processing and has been studied by having the subject face specific visual imagery tasks that are related to the use of the visual sketchpad as a component of the working memory system. Much less is known about spontaneous, free visual imagination, what circuits drive it, how and why. Motor imagery has been studied with several approaches: the neural circuits activated in the brain during performance of a movement have been compared with those involved in visually or kinaesthetically imagining performing the same movement, or in observing another person performing it. Some networks are similarly activated in these situations, although primary motor neurons are only activated during motor execution. Imagining the execution of an action seems unable to activate circuits involved in eliciting accompanying motor adjustments (such as postural adaptations) that are unconsciously (implicitly) associated to the execution of the movement. A more faithful neuronal activation is obtained through kinaesthetic motor imagination-imagining how it feels to perform the movement. Activation of sensory-motor and mirror systems, elicited by observing another person performing a transitive action, can also recruit circuits that sustain implicit motor responses that normally accompany the overt movement. This last aspect has originated the expanding and promising field of action observation therapy (AOT). The fact that the various kinds of motor imagery differentially involve the various brain networks may offer some hints on what neural networks sustain imagery in general, another activity that has an attentive component-recalling a memory, covertly rehearsing a speech, internally replaying a behaviour-and a vague, implicit component that arises from the freely flowing surfacing of internal images, not driven by intentional, conscious control.
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The adult brain retains over life endogenous neural stem/precursor cells (eNPCs) within the subventricular zone (SVZ). Whether or not these cells exert physiological functions is still unclear. In the present work, we provide evidence that SVZ-eNPCs tune structural, electrophysiological, and behavioural aspects of striatal function via secretion of insulin-like growth factor binding protein-like 1 (IGFBPL1). In mice, selective ablation of SVZ-eNPCs or selective abrogation of IGFBPL1 determined an impairment of striatal medium spiny neuron morphology, a higher failure rate in GABAergic transmission mediated by fast-spiking interneurons, and striatum-related behavioural dysfunctions. We also found IGFBPL1 expression in the human SVZ, foetal and induced-pluripotent stem cell-derived NPCs. Finally, we found a significant correlation between SVZ damage, reduction of striatum volume, and impairment of information processing speed in neurological patients. Our results highlight the physiological role of adult SVZ-eNPCs in supporting cognitive functions by regulating striatal neuronal activity.
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Proteínas de Unión a Factor de Crecimiento Similar a la Insulina , Ventrículos Laterales , Células-Madre Neurales , Proteínas Supresoras de Tumor , Animales , Humanos , Ratones , Electrofisiología Cardíaca , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/fisiología , Células-Madre Neurales/fisiología , Proteínas Supresoras de Tumor/fisiología , Ventrículos Laterales/fisiologíaRESUMEN
The effects of microgravity on frog semicircular canals have been studied by electrophysiological and morphological approaches. Reduced gravity (microG) was simulated by a random positioning machine (RPM), which continually and randomly modified the orientation in space of the anesthetized animal. As this procedure stimulates the semicircular canals, the effect of altered gravity was isolated by comparing microG-treatment with an identical rotary stimulation in the presence of normal gravity (normoG). Electrophysiological experiments were performed in the isolated labyrinth, extracted from the animals after the treatment, and mounted on a turntable. Junctional activity was measured by recording quantal events (mEPSPs) and spikes from the afferent fibers close to the junction, at rest and during rotational stimulation. MicroG-treated animals displayed a marked decrease in the frequency of resting and evoked mEPSP discharge, vs. both control and normoG (mean decrease approximately 50%). Spike discharge was also depressed: 57% of microG-treated frogs displayed no spikes at rest and during rotation at 0.1 Hz, vs. 23-31% of control or normoG frogs. Among the firing units, during one cycle of sinusoidal rotation at 0.1 Hz microG-treated units emitted an average of 41.8 + or - 8.06 spikes, vs. 77.2 + or - 8.19 in controls. Patch-clamp analysis on dissociated hair cells revealed altered Ca(2+) handling, after microG, consistent with and supportive of the specificity of microG effects. Marked morphological signs of cellular suffering were observed after microG, mainly in the central part of the sensory epithelium. Functional changes due to microgravity were reversible within a few days.
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Oído Interno/fisiología , Unión Neuromuscular/fisiología , Canales Semicirculares/fisiología , Transmisión Sináptica/fisiología , Ingravidez/efectos adversos , Anestesia , Animales , Estado de Descerebración , Electrofisiología , Potenciales Evocados/fisiología , Potenciales Postsinápticos Excitadores/fisiología , Células Ciliadas Ampollares/fisiología , Técnicas In Vitro , Fatiga Muscular/fisiología , Neuronas Aferentes/fisiología , Técnicas de Placa-Clamp , Estimulación Física , Rana esculenta , RotaciónRESUMEN
Here, we examine subjectivity and consciousness as emergent properties of the computational complexity of information processing by the brain, rather than metaphysical phenomena. While Psychology concentrates on the emergent properties and Neurobiology examines the properties of the biological substrate, Neurophysiology and Cognitive Neuroscience link the two levels by investigating the mechanisms and processes by which the functions of the brain emerge from the anatomical, cellular and network properties of the nervous system. Our purpose here is not to locate the neural structures that sustain subjectivity or other psychic functions; rather, we examine the operating modes of neurons and neural circuits: they reveal an intrinsically relational quality; sensory elaboration itself proves to be relational and self-centred, necessarily associated with the vital, hedonic, emotional relevance of each experience and external cue, and intrinsically oriented to a behavioral interaction with the latter. The hippocampus adds to this self-centred relational perspective the capability of transforming the identification and the spatial location of objects into a contextualized representation of reality. Since the hippocampus is strongly interconnected with the archaic structures that evaluate vital and hedonic relevance and generate emotional responses, the contextualized information, emotionally colored, is transformed into a comprehensive individual experience. This way, a subjective, self-centred, affectively colored perspective arises in animals due to the intrinsic properties of neuronal circuits in the brain. We conclude that neuronal network processing is strongly characterized per se by a relational and self-centred (subjective) and emotionally colored, motivationally oriented (personal) perspective. The properties and features of neural processing discussed here constitute well-established knowledge in the neuroscientific community. Yet, from a layman's perception, subjectivity still mysteriously arises in our brain due to the action of consciousness, and in epistemological and philosophical debates, the question often arises as to how consciousness may add the subjective and personal perspective to neural elaboration. The answer appears to be simple: it does not; subjectivity is already there, present ab initio in neuronal processing and not added a posteriori by some other "consciousness" function of unclear neural basis.
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Potassium-current inactivation and recovery kinetics are pivotal in sustaining dynamic processing of time-varying sensory signals in hair cells. We report a detailed analysis of K(+)-currents in isolated hair cells from the frog crista ampullaris. The single components were dissected using a novel procedure based on their differential kinetic properties: The fast IA component exhibited two processes of inactivation removal; the persistent I (KD) component (I (KV) + I (KCa)), unexpectedly displayed partial inactivation, removed by negative potentials with particularly slow, delayed kinetics. The physiological relevance of these observations was investigated by imposing sinusoidal membrane potential changes to mimic receptor response to hair bundle deflection. The excitatory phase elicited extra-currents (hysteresis) only if the off phase went sufficiently negative to remove IA inactivation. Native, resting hair cells are depolarised by receptor current; thus, voltage continuously modulates I(KD), whereas IA only transiently ensues when the receptor current vanishes (zero-current potential approximately -70 mV) and polarisation removes IA inactivation.
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Células Ciliadas Auditivas/fisiología , Potenciales de la Membrana/fisiología , Canales de Potasio/fisiología , Animales , Cadmio/farmacología , Calcio/fisiología , Cinética , Potenciales de la Membrana/efectos de los fármacos , Técnicas de Placa-Clamp , Canales de Potasio de Rectificación Interna , Rana esculentaRESUMEN
Several cellular processes depend on networks of proteins assembled at specific sites near the plasma membrane. Scaffold proteins assemble these networks by recruiting relevant molecules. The scaffold protein ERC1/ELKS and its partners promote cell migration and invasion, and assemble into dynamic networks at the protruding edge of cells. Here by electron microscopy and single molecule analysis we identify ERC1 as an extended flexible dimer. We found that ERC1 scaffolds form cytoplasmic condensates with a behavior that is consistent with liquid phases that are modulated by a predicted disordered region of ERC1. These condensates specifically host partners of a network relevant to cell motility, including liprin-α1, which was unnecessary for the formation of condensates, but influenced their dynamic behavior. Phase separation at specific sites of the cell periphery may represent an elegant mechanism to control the assembly and turnover of dynamic scaffolds needed for the spatial localization and processing of molecules.
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Movimiento Celular/fisiología , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Células COS , Línea Celular Tumoral , Membrana Celular/metabolismo , Chlorocebus aethiops , Citoplasma/metabolismo , Humanos , Proteínas del Tejido Nervioso/fisiología , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas Asociadas a Matriz Nuclear/fisiología , Proteínas de Unión al GTP rab/fisiologíaRESUMEN
The role of glutamate in quantal release at the cytoneural junction was examined by measuring mEPSPs and afferent spikes at the posterior canal in the intact frog labyrinth. Release was enhanced by exogenous glutamate, or dl-TBOA, a blocker of glutamate reuptake. Conversely, drugs acting on ionotropic glutamate receptors did not affect release; the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA-R) blocker CNQX decreased mEPSP size in a dose-dependent manner; the NMDA-R blocker d-AP5 at concentrations <200⯵M did not affect mEPSP size, either in the presence or absence of Mg and glycine. In isolated hair cells, glutamate did not modify Ca currents. Instead, it systematically reduced the compound delayed potassium current, IKD, whereas the metabotropic glutamate receptor (mGluR)-II inverse agonist, (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl)propanoic acid (LY341495), increased it. Given mGluR-II decrease cAMP production, these finding are consistent with the reported sensitivity of IKD to protein kinase A (PKA)-mediated phosphorylation. LY341495 also enhanced transmitter release, presumably through phosphorylation-mediated facilitation of the release machinery. The observed enhancement of release by glutamate confirms previous literature data, and can be attributed to activation of mGluR-I that promotes Ca release from intracellular stores. Glutamate-induced reduction in the repolarizing IKD may contribute to facilitation of release. Overall, glutamate exerts both a positive feedback action on mGluR-I, through activation of the phospholipase C (PLC)/IP3 path, and the negative feedback, by interfering with substrate phosphorylation through Gi/0-coupled mGluRs-II/III. The positive feedback prevails, which may explain the increase in overall rates of release observed during mechanical stimulation (symmetrical in the excitatory and inhibitory directions). The negative feedback may protect the junction from over-activation.
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Oído Interno/efectos de los fármacos , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Ácido Glutámico/farmacología , Células Ciliadas Auditivas/efectos de los fármacos , Sinapsis/efectos de los fármacos , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Aminoácidos/farmacología , Animales , Anuros , Ácido Aspártico/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Técnicas de Placa-Clamp , Receptores Ionotrópicos de Glutamato/antagonistas & inhibidores , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Xantenos/farmacologíaRESUMEN
BACKGROUND: The association between maternal infection and neurodevelopmental defects in progeny is well established, although the biological mechanisms and the pathogenic trajectories involved have not been defined. METHODS: Pregnant dams were injected intraperitoneally at gestational day 9 with polyinosinic:polycytidylic acid. Neuronal development was assessed by means of electrophysiological, optical, and biochemical analyses. RESULTS: Prenatal exposure to polyinosinic:polycytidylic acid causes an imbalanced expression of the Na+-K+-2Cl- cotransporter 1 and the K+-Cl- cotransporter 2 (KCC2). This results in delayed gamma-aminobutyric acid switch and higher susceptibility to seizures, which endures up to adulthood. Chromatin immunoprecipitation experiments reveal increased binding of the repressor factor RE1-silencing transcription (also known as neuron-restrictive silencer factor) to position 509 of the KCC2 promoter that leads to downregulation of KCC2 transcription in prenatally exposed offspring. Interleukin-1 receptor type I knockout mice, which display braked immune response and no brain cytokine elevation upon maternal immune activation, do not display KCC2/Na+-K+-2Cl- cotransporter 1 imbalance when implanted in a wild-type dam and prenatally exposed. Notably, pretreatment of pregnant dams with magnesium sulfate is sufficient to prevent the early inflammatory state and the delay in excitatory-to-inhibitory switch associated to maternal immune activation. CONCLUSIONS: We provide evidence that maternal immune activation hits a key neurodevelopmental process, the excitatory-to-inhibitory gamma-aminobutyric acid switch; defects in this switch have been unequivocally linked to diseases such as autism spectrum disorder or epilepsy. These data open the avenue for a safe pharmacological treatment that may prevent the neurodevelopmental defects caused by prenatal immune activation in a specific pregnancy time window.
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Corteza Cerebral/fisiología , Epilepsia/etiología , Potenciales Postsinápticos Excitadores/fisiología , Potenciales Postsinápticos Inhibidores/fisiología , Complicaciones del Embarazo/inmunología , Efectos Tardíos de la Exposición Prenatal/etiología , Ácido gamma-Aminobutírico , Animales , Técnicas de Cultivo de Célula , Modelos Animales de Enfermedad , Embrión de Mamíferos , Femenino , Ratones Endogámicos C57BL , Ratones Noqueados , Técnicas de Placa-Clamp , Embarazo , Receptores Tipo I de Interleucina-1 , Simportadores , Cotransportadores de K ClRESUMEN
The post-transductional elaboration of sensory input at the frog semicircular canal has been studied by correlating the effects of drugs that interfere with phosphorylation processes on: (i) potassium conductances in isolated hair cell and (ii) transmitter release at the cytoneural junction in the intact labyrinth. At hair cells, delayed potassium currents (IKD) undergo voltage- and time-dependent inactivation; inactivation removal requires ATP, is sensitive to kinase blockade, but is unaffected by exogenous application of cyclic nucleotides. We report here that forskolin, an activator of endogenous adenylyl cyclase, enhances IKD inactivation removal in isolated hair cells, but produces an overall decrease in IKD amplitude consistent with the direct blocking action of the drug on several families of K channels. In the intact labyrinth, forskolin enhances transmitter release, consistent with such depression of K conductances. Kinase blockers - H-89 and KT5823 - have been shown to reduce IKD inactivation removal and IKD amplitude at isolated hair cells. In the labyrinth, the effects of these drugs on junctional activity are quite variable, with predominant inhibition of transmitter release, rather than the enhancement expected from the impairment of K currents. The overall action of forskolin and kinase inhibitors on K conductances is similar (depression), but they have opposite effects on transmitter release: this indicates that some intermediate steps between the bioelectric control of hair cell membrane potential and transmitter release are affected in opposite ways and therefore are presumably regulated by protein phosphorylation.
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Carbazoles/farmacología , Colforsina/farmacología , Células Ciliadas Ampollares/efectos de los fármacos , Isoquinolinas/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Sulfonamidas/farmacología , Potenciales de Acción/efectos de los fármacos , Proteínas Anfibias/metabolismo , Animales , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Células Ciliadas Ampollares/metabolismo , Potenciales Postsinápticos Miniatura/efectos de los fármacos , Técnicas de Placa-Clamp , Potasio/metabolismo , Canales de Potasio/metabolismo , Rana esculenta , Técnicas de Cultivo de TejidosRESUMEN
Human serum albumin (HSA), the most prominent protein in plasma, is best known for its extraordinary ligand binding capacity. The three homologous domains of HSA (labeled I, II, and III), each in turn composed of two subdomains (named A and B), give rise to the three-dimensional structure of HSA. This flexible structural organization allows the protein structure to adapt to a variety of ligands. As conformational adaptability of HSA extends well beyond the immediate vicinity of the binding site(s), cooperativity and allosteric modulation arise among binding sites; this makes HSA similar to a multimeric protein. Although kinetic and thermodynamic parameters for ligand binding to HSA calculated by quantitative structure-activity relationship models are in excellent agreement with those obtained in vitro, cooperative and allosteric equilibria between different binding sites and competition between drugs or between drugs and endogenous ligands make difficult the interpretation of HSA binding properties in vivo. Binding of exogenous and endogenous ligands to HSA appears to be relevant in drug therapy and management. Here, the allosteric modulation of drug binding to HSA is briefly reviewed.
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Preparaciones Farmacéuticas/metabolismo , Albúmina Sérica/metabolismo , Regulación Alostérica , Humanos , Modelos Moleculares , Unión Proteica , Albúmina Sérica/químicaRESUMEN
Up to now, baclofen (a GABA(B) receptor agonist) has been used for the treatment of severe spasticity unresponsive to oral antispasmodics. Although in humans it is usually administered at 2 mg/ml, the dosage to be used in the treatment of other diseases is unknown. For this reason, it is important to determine the safe maximum dosage and toxicity at the clinically used concentration. Primary cortical neurons represent a useful model to test the safety of baclofen. We performed a colorimetric assay (MTT test) as well as electron microscopy investigations, to determine neuronal survival after the treatment with baclofen at a concentration of 2 and 4 mg/ml. Our results demonstrated that, in our experimental model, neither concentration affected neuronal survival. Considering the above results, we can conclude that at the used concentrations, this drug is safe and its clinical use should be encouraged.
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Baclofeno/uso terapéutico , Agonistas del GABA/uso terapéutico , Relajantes Musculares Centrales/uso terapéutico , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Colorimetría , Interpretación Estadística de Datos , Femenino , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Neuronas/ultraestructura , Embarazo , Ratas , Sales de Tetrazolio , TiazolesRESUMEN
Human serum albumin (HSA) is best known for its extraordinary ligand binding capacity. HSA has a high affinity for heme and is responsible for the transport of medium and long chain fatty acids. Here, we report myristate binding to the N and B conformational states of Mn(III)heme-HSA (i.e. at pH 7.0 and 10.0, respectively) as investigated by optical absorbance and NMR spectroscopy. At pH 7.0, Mn(III)heme binds to HSA with lower affinity than Fe(III)heme, and displays a water molecule coordinated to the metal. Myristate binding to a secondary site FAx, allosterically coupled to the heme site, not only increases optical absorbance of Mn(III)heme-bound HSA by a factor of approximately three, but also increases the Mn(III)heme affinity for the fatty acid binding site FA1 by 10-500-fold. Cooperative binding appears to occur at FAx and accessory myristate binding sites. The conformational changes of the Mn(III)heme-HSA tertiary structure allosterically induced by myristate are associated with a noticeable change in both optical absorbance and NMR spectroscopic properties of Mn(III)heme-HSA, allowing the Mn(III)-coordinated water molecule to exchange with the solvent bulk. At pH = 10.0 both myristate affinity for FAx and allosteric modulation of FA1 are reduced, whereas cooperation of accessory sites and FAx is almost unaffected. Moreover, Mn(III)heme binds to HSA with higher affinity than at pH 7.0 even in the absence of myristate, and the metal-coordinated water molecule is displaced. As a whole, these results suggest that FA binding promotes conformational changes reminiscent of N to B state HSA transition, and appear of general significance for a deeper understanding of the allosteric modulation of ligand binding properties of HSA.
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Regulación Alostérica , Hematoporfirinas/metabolismo , Metaloporfirinas/metabolismo , Ácido Mirístico/metabolismo , Albúmina Sérica/metabolismo , Sitio Alostérico , Sitios de Unión , Humanos , Concentración de Iones de Hidrógeno , Unión Proteica , Conformación Proteica , Análisis Espectral , Agua/químicaRESUMEN
At the frog semicircular canals, the afferent fibers display high spontaneous activity (mEPSPs), due to transmitter release from hair cells. mEPSP and spike frequencies are modulated by stimulation that activates the hair cell receptor conductance. The relation between receptor current and transmitter release cannot be studied at the intact semicircular canal. To circumvent the problem, we combined patch-clamp recordings at the isolated hair cell and electrophysiological recordings at the cytoneural junction in the intact preparation. At isolated hair cells, the K channel blocker tetraethylammonium (TEA) is shown to block a fraction of total voltage-dependent K-conductance (IKD) that depends on TEA concentration but not on membrane potential (V m). Considering the bioelectric properties of the hair cell, as previously characterized by this lab, a fixed fractional block of IKD is shown to induce a relatively fixed shift in V m, provided it lies in the range -30 to -10 mV. The same concentrations of TEA were applied to the intact labyrinth while recording from single afferent fibers of the posterior canal, at rest and during mechanical stimulation. At the peak of stimulation, TEA produced increases in mEPSP rate that were linearly related to the shifts produced by the same TEA concentrations (0.1-3 mM) in hair cell V m (0.7-5 mV), with a slope of 29.8 Hz/mV. The membrane potential of the hair cell is not linearly related to receptor conductance, so that the slope of quantal release vs. receptor conductance depends on the prevailing V m (19.8 Hz/nS at -20 mV; 11 Hz/nS at -10 mV). Changes in mEPSP peak size were negligible at rest as well as during stimulation. Since ample spatial summation of mEPSPs occurs at the afferent terminal and threshold-governed spike firing is intrinsically nonlinear, the observed increases in mEPSP frequency, though not very large, may suffice to trigger afferent spike discharge.
RESUMEN
The effects of endogenous and exogenous Ca(2+) buffers on Ca(2+) current kinetics have been investigated by patch clamp in hair cells mechanically isolated from frog semicircular canals. This preparation displays at least three different Ca(2+) channel types: transient currents flow through a drug-resistant channel ("R1"), while non-inactivating channels sustain a steady, plateau current comprised of a large L component and a small drug-resistant fraction ("R2"). In the perforated-patch condition a large and stable Ca(2+) current was recorded, with all three components. In whole-cell, a buffer-free pipette solution did not prevent a complete Ca(2+) response. The size of the transient and plateau current fractions were greatly reduced, but the ratio between the two fractions, as well as the activation, inactivation and deactivation kinetics, were substantially unmodified. Current amplitude partially recovered with 5 mM EGTA in the pipette solution. With 50 mM EGTA all the kinetic parameters were slowed down and the transient component, but not the plateau component, markedly increased in size. Response kinetics slowed down even more with 30 mM Cs-BAPTA and the Ca(2+) waveform was substantially modified. The transient component was very large and inactivated slowly; the remaining very small plateau fraction deactivated along a slow, single exponential time. Under this condition nifedipine (10 microM) produced a great reduction of the transient current, leaving plateau and deactivation phase unaltered. This suggests that only R2 channels were still active at the end of the test and that the minor remaining transient component flowed through slowly but completely inactivating R1 channels. These results confirm the presence of several channel types in semicircular canal receptors, at difference with cochlear hair cells, and highlight a dramatic alteration of L-type channel behavior when intracellular Ca(2+) buffers are sufficiently concentrated and fast to interfere with rapid and local changes in Ca(2+) levels.